Bacterial infections pose serious threats to human health. Furthermore, viral infections such as the flu are frequently accompanied by bacterial infections which is often a deadly combination. Due to the development of resistance, the options for treating infections have dwindled substantially. This resistance is in large part due to the bacterial expression of beta-lactamases that degrade beta-lactam antibiotics including penicillins, cephalosporins, and the "last resort" carbapenem antibiotics. An alternative approach for treating p-lactam resistant gram-negative infections is co-administering a p-lactamase inhibitor in addition to penicillin-like antibiotics. Regrettably, p-lactamases have also evolved an inhibitor-resistant phenotype able to overcome this treatment option. The p-lactamase variants that hydrolyze these inhibitors are called inhibitor resistant [3-lactamases, those that hydrolyze cephalosporins are called extended-spectrum p-lactamases (ESBLs), and those that hydrolyze carbapenems are known as carbapenemases. The overarching goals of this proposal are to understand the structural basis of the phenotypes of ESBL-, carbapenemase-, and inhibitor resistant p-lactamases, and to develop novel inhibition strategies. Our structure-function studies involve a novel synergy between X-ray and Raman crystallography and this innovative inter-disciplinary approach allows us to identify and track reaction intermediates inside crystals prior to X-ray analysis and provides a unique advantage to accomplish our Aims. Aim 1: To further improve our novel designed beta-lactamase inhibitor SA2-13 by modifying the overall charge and carboxyl linker to improve uptake and trans-enamine stabilization. Aim 2: To test the hypothesis that the changes in or near D179 which are present in ESBL's SHV-6, -8, and -24 have evolved to hydrolyze ceftazidime by shifting the omega loop thereby extending the active site to accommodate ceftazidime. Aim 3: To test the hypothesis that class A carbapenemases such as KPC-2 have adopted a shallower active site and flexible catalytic 870 side chain to efficiently hydrolyze carbapenems. Aim 4: To test the hypothesis that inhibitors that either can carry out bi-cyclization (such as LN1-255) or fra/is-enamine inhibitors (SA2-13) are capable of forming stable inhibitory complexes with inhibitor-resistant class A and inhibitor-insensitive class D p-lactamases. The impact of beta-lactamase mediated antibiotic resistance on human health is enormous, costing billions of dollars in health care costs. Detailed understanding is needed and our targeted structural knowledge will provide for molecular insights into cases such as the recent outbreaks of KPC mediated carbapenem-resistant K. pneumoniae in New York. These resistance insights will lead to new therapeutic approaches and our goal is to study and develop new broad-spectrum beta-lactamase inhibitors.